The current studies focus on the signaling pathways controlling myelination. Transgenic mice that overexpress constitutively active Akt (protein kinase B) in oligodendrocytes produce increased amounts of myelin. Furthermore, there are effects on the neurons in these mice. The current studies focus on identifying the signaling pathways by which Akt regulates myelination, and the impact of Akt expression in oligodendrocytes on neurons. The hypothesis to be tested is that Akt signaling directly regulates oligodendrocyte differentiation and myelination. This will be investigated initially by studying the signaling pathways and gene expression changes that occur in these mice. Thus, the first aim will focus on the signaling pathways downstream of Akt that regulate CMS myelination. Initial studies will investigate the activation/inactivation state of a series of Akt phospho-substrates, including transcription factors, mTOR and other known Akt substrates. Our studies demonstrate that Akt overexpression in these mice induces the MAP kinase pathway. We will therefore investigate this pathway as well, focusing on Erk1/2 signaling. The main focus of this aim is on mRNAs and proteins that regulate the hypermyelination phenotype. We will investigate whether this is exclusively controlled by an inability to stop myelinating, or if there is also an impact on differentiation. Thus, we will study whether and how Akt overexpression impacts oligodendrocyte differentiation, in addition to impacting the long-term myelination process. We will investigate how Akt signaling drives CMS myelination as assessed by expression of known oligodendrocyte lineage and differentiation markers, with particular interest in the transcription factors regulating myelination. Putative major Akt substrates controlling myelination or oligodendrocyte differentiation will be confirmed by over- expressing or knocking down expression of these proteins in cultured oligodendrocytes. The second aim of the proposal focuses on the effect of elevated Akt expression and increased myelination on developing neurons. Studies focus on axonal development in optic nerve, investigating morphological maturation and organization of nodes of Ranvier. Our preliminary data demonstrate strong interactions between oligodendrocytes overexpressing Akt and developing axons. Interactions of oligodendrocytes and neurons will be studied in culture, using cells obtained from PLP-Akt-DD mice. The importance of these studies is that identifying the crucial Akt substrate(s) that regulates myelination may lead to therapeutic approaches to enhance remyelination in multiple sclerosis.